Non-alcoholic Fatty Liver Disease and Gallstones: A Systematic Review

Non-alcoholic fatty liver disease (NAFLD) is steatosis of the liver that resembles alcohol-induced liver injury but is a metabolic disorder. Most patients are obese with increased triglyceride levels due to increased intake of fatty food, which can cause excess fat to build up in the liver. At the same time, continuous ingestion of fatty foods can lead to gallstones (GS) due to the overproduction of cholesterol. NAFLD and GS have been seen to coincide, and there might be a relationship between them. This systematic review analyzes the incidence of NAFLD and GS to determine a bidirectional relationship. A comprehensive literature review was done using ProQuest, PubMed, and ScienceDirect, and included only experimental studies and meta-analyses. The search included the keywords ‘gallstones and non-alcoholic fatty liver disease’ and ‘cholelithiasis and non-alcoholic fatty liver disease’. Our initial search included 10,665 articles and was narrowed down to 19 through extensive inclusion and exclusion criteria. There is a bidirectional relationship between the incidence of NAFLD and GS, where an increase in either can lead to an increase in the other. Both NAFLD and GS share similar risk factors leading to the development of each disease. On average, there’s an increase in the prevalence of gallstones in NAFLD patients, and patients with GS were also more likely to have NAFLD. There was a prevalence of NAFLD in those with asymptomatic gallstones as well, indicating that the risk factors are crucial in the development of both. As a result, some research is determining whether an evaluation of the liver should be routine during cholecystectomy due to the increased risk of developing NAFLD.


Non-alcoholic fatty liver disease
Nonalcoholic fatty liver disease (NAFLD) is a condition diagnosed with liver imaging, specifically ultrasound, when there is an accumulation of fat, a disorder known as steatosis.This fat accumulation makes up more than 5% of the weight of the liver, in the absence of alcohol as the cause but resembles alcohol-induced liver injury [1].NAFLD is categorized as a metabolic disorder due to the role of hormones, nutrition, and genetics in its pathogenesis [2].Most patients with NAFLD are obese and therefore have an increased serum triglyceride level.As in obesity, there is an increased intake of fatty foods with reduced energy output [3].Furthermore, in patients with insulin resistance, there is decreased storage of lipids in adipose tissue, which leads to increased lipolysis, allowing excess free fatty acids to enter hepatocytes resulting in steatosis; additionally, the free fatty acids in the liver undergo oxidation leading to further liver damage [1].Therefore, based on the study by Pouwels et al., the first step of acquiring NALFD is the accumulation of lipids in hepatocytes which eventually leads to insulin resistance, leading to the second step, which is oxidative stress resulting in further liver damage and inflammation [2].

Gallstones
Gallstones (GS) are precipitations in the gallbladder, an organ located beneath the liver that stores, concentrates, and releases bile into the small intestines [4].GS are comprised of cholesterol, bilirubin, and calcium salts with some proteins [5].GS are normally asymptomatic, but symptoms arise when the stone obstructs the biliary passage leading to abdominal pain called biliary colic [4].Most GS are cholesterol GS, but there are also black-pigmented GS and brown-pigmented GS which primarily consist of calcium hydrogen bilirubinate [5].Cholesterol GS are created when there is excessive secretion of cholesterol into bile by hepatocytes or impaired emptying or hypomotility of the gallbladder [6].Excess cholesterol cannot dissolve in the bile and precipitates as crystals that eventually form stones [5].Furthermore, if the gallbladder has impaired motility and cannot empty the bile, the bile becomes concentrated and begins to harden to form stones [6].The aim of this paper is to assess the relationship and prevalence of GS with NAFLD due to their similarities in pathogenesis.
An exhaustive and extensive literature search was done using PubMed, ProQuest, and ScienceDirect databases from January 1st, 2002, to December 31st, 2022.Keywords included 'gallstones and nonalcoholic fatty liver disease' and 'cholelithiasis and nonalcoholic fatty liver disease'.The electronic search focused on peer-reviewed, experiment publications that were in line with the scope of this paper.Publications not written in English, published prior to 2002 and duplicates were excluded from the screening process.Once the publications were found, three independent co-authors examined the information.The publications found in the search were examined based on their full-text accessibility, study type, title, and abstracts.The initial search of the three databases resulted in 10,665 publications.The selected publications were further narrowed down based on keyword specifics and the overview provided by the abstracts.A total of 19 publications were found to be within scope, according to the following criteria.

Inclusion Criteria
The following inclusion criteria were used: publications written in English, conducted on humans, published between 2002 and 2022, focused on the prevalence and incidence of gallstones and NAFLD together, fulltext inclusive of both subscription and non-subscription articles, and peer-reviewed with variations between cohort, case-control, observation studies, and meta-analyses.

Exclusion Criteria
Exclusion criteria included case series/reports, systematic reviews, and review articles.All non-full-text publications and duplicates were also excluded.The procedure of inclusion and exclusion of the publications is drawn out in Figure 1.

Bias
All studies were assessed for bias.The studies showed a medium risk of bias as most studies disclosed their protocols and methods and focused on databases.The individual risk of bias was evaluated using the Grading of Recommendation, Assessment, Development, and Evaluations (GRADE) tool, which assessed flaws like imprecision, indirectness, and publications.
The algorithm for this literature review was done as described in the PRISMA statement [7].

Review Results
A total of, 10665 publications were found; 215 were from PubMed, 1842 were from ScienceDirect, and 8608 were from ProQuest.Among the exclusions, 2302 were duplicate publications, and four were published before 2002.This resulted in 2306 publications being excluded during the automatic screening process, leading to 8359 publications for manual screening.Publications were manually screened based on title, study type, abstract, and availability, resulting in 55 articles being checked for eligibility.Ultimately 19 articles were used.
Due to the similarities in pathogenesis, there should be a corresponding incidence rate between GS and NAFLD.It was found that there is a bidirectional relationship between GS and NAFLD with a significant association.Biomarkers of NAFLD include macroglobulins, haptoglobulins, total bilirubins, transferases, and transaminases.A single marker alone of NAFLD increases the chance of developing GS.Elderly individuals and females are more likely to develop GS with an underlying NAFLD diagnosis.Research is unsure whether the trend is the same with asymptomatic gallstones.There is also a greater risk of NAFLD for those who've undergone a cholecystectomy.

Discussion
There is a bidirectional relationship between the prevalence of NAFLD and gallstones, indicating an increased risk in both [8].There is a positive correlation between NAFLD and GS in that the risk factors for developing both diseases were similar [9,10].There is a significant association between NAFLD and GS, even when accounting for heterogeneity [11,12].The presence of gallstones and previous cholecystectomy increased significantly in NAFLD patients [13].Koller et al. observed that 19% of patients have both gallstones and one marker of NALFD [8].A patient who has at least one marker of fatty liver disease, whether it is an increase in macroglobulins, haptoglobulins, total bilirubins, transferases, or aminotransaminases, had nearly a double prevalence of gallstones compared to patients with no markers (p < 0.0001) [8].The development of GD was positively correlated with the grade of NAFLD (p < 0.001) [14,15].

Prevalence of Gallstones and NAFLD
The prevalence of GS in NAFLD varied between studies but averaged about 15% [10,14,16,17].One of the mechanisms underlying this occurrence may be due to NAFLD leading to excessive cholesterol accumulation and altered cholesterol metabolism resulting in the development of GS [18].Patients with GS were more likely to have NAFLD by 6.85 times (p < 0.01) compared to those with cholecystectomy, which were 2.14 times more likely [19].Subjects with GS had higher rates of metabolic syndrome, hypertension, medication use for diabetes, dyslipidemia, and high-grade NAFLD than those without [15].GS was independently associated with NAFLD when accounting for age, sex, and metabolic profile [13].Ahmed et al. did observe that those who had a family history of GS and those who had GS with NAFLD had a first-degree relative with GS (p < 0.034) [20].Kichloo et al. observed that Caucasian patients with NAFLD had a higher prevalence of GS which was not replicable in other racial groups [19].GS was significantly associated with older patients, and this trend continued with those who also had NAFLD [10,13,17,19,21,22].This may be due to reduced serum highdensity lipoprotein (HDL) as HDL is needed to regulate cholesterol secretion (Hung et al., 2020).Female patients with NAFLD were also more likely to develop GS complications with an increased prevalence [11,14,17,19,23].However, one study found no statistically significant association with the development of GS [18].

Prevalence of Asymptomatic Gallstones and NAFLD
There is debatable research on whether asymptomatic GS are associated with NAFLD or not, as most people are unaware that they have GS.The prevalence of NAFLD was 59% in patients with asymptomatic gallstones compared to 46.7% in those without gallstones [21,24].A significant association exists between NAFLD and increased incidence of asymptomatic GS after accounting for metabolic risk factors (p = 0.006) [24].This association is stronger in females and patients who were < 50 years of age (p < 0.0001 for both) [24].However, one study found that asymptomatic GS was negatively associated with NAFLD [25].

Prevalence of Cholecystectomy and NAFLD
Several studies evaluated the relationship between cholecystectomy and NAFLD and whether routine liver analysis should be standard for these patients.Hajong found that of 200 patients undergoing cholecystectomy, 138 had negative testing for NAFLD, 39 were borderline or inconclusive, and 23 had definitive tests [26].Patients who had cholecystectomy had a 35% increase in the risk of developing NAFLD (Kwak et al. [13]).Vice versa, cholecystectomy's prevalence was significantly higher in patients with NAFLD [25].Additionally, there was a significantly stronger association between NAFLD and the incidence of cholecystectomy in women than in men (p < 0.033) [18].The increased risk of developing NAFLD may be because cholecystectomy leads to decreased fibroblast growth factor 19, which affects cholesterol metabolism, causing triglycerides to accumulate in the liver [18].The grounds for routine liver biopsy during or after cholecystectomy for GS are substantiated due to the high occurrence of NAFLD in these patients, which can escape detection for an extended period [9].Some limitations of this article arose because of the limited number of studies analyzing the relationship between GS and NAFLD.There's research about the varying pathogenesis that may correlate to a relationship, but these are the only articles that address this relationship.This article also excluded animal studies that could elucidate the actual relationship displayed.A summary of the discussion/reviewed findings is presented in Table 1.

Author Country
Design &

Study Population
Findings Conclusion Of the sample of 441 patients, 54 patients (12.2%) had gallstone disease (GD).77% of the patients that had GD had undergone cholecystectomy for symptomatic GD.The patients who were GD+ were found to be older, had higher BMIs, and were more likely to be female.It was also found that patients who had GD did not have a higher risk for fibrosis or non-alcoholic steatohepatitis on histology.
The There was a significantly higher prevalence of gallstones among patients with NAFLD compared to those without (p < 0.0001).Gallstones are independent predictors of NAFLD (OR = 1.77).56% of patients with gallstones had NAFLD compared to 33% who did not.NAFLD is also an independent predictor of gallstones (OR = 1.92).
Metabolic risk factors such as BMI, triglycerides, diabetes, and total cholesterol concentrations were also independent predictors of NAFLD and gallstones Gallstones are an independent risk factor of NAFLD and vice versa along with metabolic risk factors.There is an association between gallstones and NAFLD (p = 0.047).After adjustment for sex, it was found that specifically in females there was an association between NAFLD and gallstones (p = 0.001).
NAFLD is associated with gallstones with a stronger association apparent in females compared to males.
There was no significant difference in the rate of gallstones between subjects with and without NAFLD before and after case-control matching (p = Due to higher proportion of

Conclusions
Based on this research, there is a bidirectional relationship between the development of GS and NAFLD.
Patients with NAFLD have a significantly higher prevalence of GS throughout their disease.This correlation falls back to the pathogenesis of both NAFLD and GS, which have origins in dysfunctional cholesterol and fat metabolism.Patients more likely to develop GS concurrently tend to be Caucasian, elderly, and female.Going further into the association, we questioned whether this relationship occurred with patients suffering from asymptomatic GS.Research, however, is still split on whether there's an association with NAFLD.There's also a significant association between the incidence of cholecystectomy and NAFLD.The increased risk of developing NAFLD may be due to a decrease in fibroblast growth factor 19, another alteration to cholesterol metabolism.Further research should be conducted to determine if routine testing for the possible association should be done during a typical workup due to their hand-in-hand relationship.